Liquid chromatographic determination of amoxicillin and its metabolites in human urine by postcolumn degradation with sodium hypochlorite

Abstract

A high-performance liquid chromatographic method has been developed for the determination of amoxicillin (I) and its metabolites [(5 R,6 R)-amoxicilloic acid (II), the (5 S,6 R) epimer (III), and and (2 R)-piperazine-2′,5′-dione (IV)] in human urine. They were separated from the background components of urine on a reversed-phase C 18 column using sodium heptylsulphonate as an ion-pairing agent and methanol as an organic mobile phase modifier. The eluent was led to the postcolumn degradation with 1.5 M sodium hydroxide plus 0.02% sodium hypochlorite solution at ambient temperature. The degradation product(s) of each compound was detected at 270 nm. The proposed method permits detection of I, II, III, and IV down to 1 μg/ml in neat urine samples. At a concentration of 5 μg/ml of each compound, within-and between-run precisions (relative standard deviation) were 1.12–5.79 and 0.90–2.70%, respectively. The urinary levels of I and its metabolites were determined by the proposed method after administration of I to humans.